The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• gating iontového kanálu genetika   MeSH
• genetické vektory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• liposomy chemie   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• reportérové geny MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vápníková signalizace * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Úvod: Febrilní záchvaty (febrile seizures, FS) jsou nejčastějším typem epileptických záchvatů v dětském věku. Generalizovaná epilepsie s febrilními záchvaty plus (generalized epilepsy with febrile seizures plus, GEFS+) je autozomálně dominantně (AD) dědičný syndrom definovaný výskytem FS (i po 6. roce věku, tj. febrilní záchvaty plus, FS+), které asi u třetiny pacientů přecházejí v afebrilní epileptické záchvaty. Asi u 70 % pacientů se syndromem GEFS+ jsou jediným projevem FS nebo FS+. Syndrom GEFS+ je zhruba v 10 % případů způsoben mutací v genu SCNIA pro αl podjednotku sodíkového napěťově řízeného neuronálního kanálu. Cil práce: Vzhledem ke známému molekulárnímu podkladu a častému výskytu fenotypu FS mezi probandy se syndromem GEFS+jsme si vytyčili za úkol ozřejmění podílu mutací v genu SCNIA mezi probandy se sporadickými FS nebo familiárními AD dědičnými FS. Materiál a metodika: Do projektu bylo zařazeno 50 probandů s výskytem FS a 50 zdravých kontrol. Celkem byla odebrána anamnestická data 405 osob (probandů a rodinných příslušníků). Mutační analýza všech 26 exonů genu SCNIA byla provedena pomocí SSCP (Single-stranded conformation polymorphism) analýzy. Výsledky: Tricet pět probandů bylo z rodin s AD přenosem FS. 15 probandů mělo FS s nefamiliárním výskytem. U žádného z 50 vyšetřených probandů nebyla nalezena mutace v genu SCNIA. Závěry: Nebyly zjištěny významné rozdíly v klinických parametrech mezi probandy s izolovaným a familiárním výskytem FS. Mutace v genu SCNIA nejspíše není významným etiologickým faktorem vzniku FS.

Introduction: Febrile seizures (FS) are the most common form of childhood seizures. Generalized epilepsy with febrile seizures plus (GEFS+) is an epileptic syndrome with autosomal dominant (AD) inheritance. GEFS+ is characterized by the incidence of FS persisting beyond the age of 6 years (defined as FS plus, FS+), which are followed in about one-third of patients by afebrile epileptic seizures. FS or FS+ are the only phenotypes described in around 70 % of GEFS+ probands. In approximately 10 % of cases GEFS+ syndrome is caused by mutations in SCNIA gene (coding for αl subunit of neuronal voltage-gated sodium channel). The aim of the study: Due to well-known genetic background and frequent occurrence of FS phenotype in the GEFS+ patients the aim was to analyse the proportion of SCNIA mutations in patients with sporadic FS and AD inherited FS. Materials and methods: 50 FS probands and 50 healthy control subjects were included in the study. Clinical data from the total of 405 subjects were analysed. SSCP (Single-stranded conformation polymorphism) mutational analysis of all 26 exons of SCNIA gene was performed. Results: 35 FS probands came from families with AD inheritance. 15 probands had sporadic FS. No mutations in SCNIA gene were found. Conclusion: No significant clinical differences between probands with sporadic and familial FS were revealed. The SCNIA gene mutation is probably an insignificant etiological factor in FS.

• MeSH
• anamnéza MeSH
• diagnostické techniky molekulární MeSH
• epilepsie diagnóza   genetika   MeSH
• febrilní křeče diagnóza   etiologie   MeSH
• fenotyp MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• mutace MeSH
• polymorfismus konformace jednovláknové DNA MeSH
• sodíkové kanály MeSH
• Check Tag
• lidé MeSH

The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.

• MeSH
• aktivace transkripce genetika   MeSH
• arginin metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Drosophila melanogaster MeSH
• gating iontového kanálu genetika   MeSH
• genomika MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádory metabolismus   MeSH
• nemoci svalů metabolismus   MeSH
• protein ORAI1 genetika   metabolismus   MeSH
• protein STIM1 genetika   metabolismus   MeSH
• sekundární struktura proteinů genetika   MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)-based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients. PROCEDURE: We focused at the design of standardized and reproducible RD monitoring in ALL. RD was investigated by a uniform gating strategy, which was designed internationally and tested in one center by Ig/TCR rearrangements. RESULTS: For each gate, positivity cutoff value was assigned using quantification of non-leukemic background. Comparing to Ig/TCR at 0.1% level, 80 of 103 specimens were correctly diagnosed by FC. The predictive value of FC RD at day 15 was then analyzed. In B lineage ALL, day 15 FC significantly correlated with Ig/TCR results at day 33 and/or week 12 (P < 0.01). No significant correlation was found in T lineage ALL. CONCLUSIONS: Thus, FC with preset uniform gating at day 15 predicts PCR-detectable MRD in B precursor ALL. Presented data may be used to define new polychromatic cytometric diagnostics of MRD including semiautomatic assessment. Pediatr Blood Cancer 2010; 54:62-70. (c) 2009 Wiley-Liss, Inc. Copyright 2009 Wiley-Liss, Inc.

• MeSH
• dítě MeSH
• genová přestavba MeSH
• indukce remise MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• pre-B-buněčná leukemie diagnóza   genetika   MeSH
• prognóza MeSH
• průtoková cytometrie MeSH
• receptory antigenů T-buněk genetika   MeSH
• reziduální nádor diagnóza   genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: Most mutations in the myelin protein zero gene (MPZ) typically cause a severe demyelinating/dysmyelinating neuropathy that begins in infancy or an adult-onset axonal neuropathy. Axonal degeneration in the late-onset H10P mutation may be caused by the disruption of axoglial interaction. OBJECTIVE: To evaluate sural nerve biopsy samples from a patient with early-onset Charcot-Marie-Tooth disease type 1B caused by an arg69-to-cys (R69C) mutation. Design and PARTICIPANTS: Biopsies of sural nerves were performed 20 years apart in a patient with an R69C mutation (early onset). In addition, peripheral nerves were obtained from autopsy material from a patient with a T95M mutation (late onset). These nerves were analyzed using light microscopy of semithin sections, teased nerve fiber immunohistochemical analysis, electron microscopy, and immunologic electron microscopy. MAIN OUTCOME MEASURES: Pathological changes in sural nerve. RESULTS: Both R69C biopsy samples showed prominent demyelination and onion bulb formation, unlike the late-onset T95M mutation, which showed primarily axonal degeneration with no onion bulbs. The sural biopsy sample obtained 20 years earlier from the R69C patient showed minimal difference from the present sample, consistent with the lack of clinical progression during the 2 decades. Teased fiber immunohistochemical analysis of R69C revealed voltage-gated sodium channel subtype 1.8 expressions at the nodes of Ranvier around the areas of segmental demyelination. Internodal length in all R69C nerve fibers was invariably short (>94% of all internodes are <150 mum). CONCLUSIONS: Morphologic abnormalities in this early-onset R69C neuropathy were severe in childhood but progressed very slowly after adolescence. The switch to voltage-gated sodium channel subtype 1.8 expression at the nodes may provide clues into the pathogenesis of this case of early-onset neuropathy, and the short internodes may contribute to the extremely slowed conduction velocities in this case (<10 m/s).

• MeSH
• axony patologie   MeSH
• Charcotova-Marieova-Toothova nemoc genetika   patofyziologie   MeSH
• elektronová mikroskopie MeSH
• gating iontového kanálu fyziologie   MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie MeSH
• iontové kanály fyziologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• myelinová pochva * fyziologie   MeSH
• myelinový P0 protein * genetika   MeSH
• nervová vlákna patologie   MeSH
• nervus suralis patologie   MeSH
• nervus ulnaris patologie   MeSH
• progrese nemoci MeSH
• senioři nad 80 let MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny SecA metabolismus   MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• translokační kanály SEC chemie   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH

The neurotransmitter serotonin has been critically implicated in the pathogenesis of several mental disorders. The serotonin transporter (5-HTT) is a key regulator of serotonergic neurotransmission and its genetic variability is associated with increased risk of psychopathology. One well known polymorphic locus in the 5-HTT gene affecting its expression is a tandem repeat in the promoter region (5-HTTLPR). It has been reported that 5-HTT is functionally coupled with the neuronal nitric oxide synthase (NOS1 or nNOS), an enzyme catalyzing the production of nitric oxide (NO). We have previously demonstrated that a tandem repeat polymorphism in the promoter of NOS1 exon 1f (Ex1f-VNTR) is associated with sensorimotor gating, a marker of inhibitory processing and a well established endophenotype of several neuropsychiatric disorders. Here we investigated the combined genetic effects of NOS1 Ex1f-VNTR and 5-HTTLPR on sensorimotor gating, measured by prepulse inhibition (PPI) of the acoustic startle reflex, in 164 healthy adults. We found no evidence for the interaction between NOS1 Ex1f-VNTR and 5-HTTLPR on PPI. PPI was associated with NOS1 Ex1f-VNTR, but not 5-HTTLPR. Our data suggest that while NOS1 plays a role in sensorimotor gating, the nitrergic pathway of gating regulation does not involve the action of 5-HTT.

• MeSH
• dospělí MeSH
• exony MeSH
• fenotyp MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé MeSH
• membránové transportní proteiny pro serotonin genetika   MeSH
• minisatelitní repetice MeSH
• mladý dospělý MeSH
• prepulsní inhibice genetika   MeSH
• promotorové oblasti (genetika) MeSH
• senzorimotorický kortex fyziologie   MeSH
• synthasa oxidu dusnatého, typ I genetika   MeSH
• úleková reakce genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873-4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Nav1.6 voltage-gated sodium and Cav3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Nav1.6 and Cav3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs.

• MeSH
• aktivační mutace MeSH
• bodová mutace MeSH
• duplikace genu MeSH
• epilepsie tonicko-klonická genetika   MeSH
• gating iontového kanálu genetika   fyziologie   MeSH
• genetická predispozice k nemoci MeSH
• lidé MeSH
• missense mutace MeSH
• mnohočetné abnormality genetika   MeSH
• napěťově řízený sodíkový kanál, typ 6 genetika   fyziologie   MeSH
• novorozenec MeSH
• refrakterní epilepsie genetika   MeSH
• rodokmen MeSH
• skolióza genetika   MeSH
• vápníkové kanály - typ T genetika   fyziologie   MeSH
• vývojové poruchy u dětí genetika   MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• buněčná membrána metabolismus   MeSH
• gating iontového kanálu MeSH
• iontový transport MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• neurony cytologie   metabolismus   MeSH
• proteinové domény MeSH
• regulace genové exprese MeSH
• sekundární struktura proteinů MeSH
• termodynamika MeSH
• vápník metabolismus   MeSH
• vápníkové kanály - typ T genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• konzervovaná sekvence MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH